Kit and method for detecting methylation level of liver cancer risk gene TSPYL5

A methylation and kit technology is applied in the field of kits for detecting the methylation level of liver cancer risk gene TSPYL5, which can solve the problems of time-consuming, capital and energy-consuming, expensive and cumbersome process.

Active Publication Date: 2015-07-08
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method consumes time, money, and energy. At least 10 or more clones must be sequenced to obtain reliable data. A large number of clones and plasmid extraction and sequencing are required. The process is cumbersome and expensive.
Therefore, it is difficult to achieve high-throughput automated detection in the clinic, but it can be used as a comparison method for new methods of methylation detection

Method used

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  • Kit and method for detecting methylation level of liver cancer risk gene TSPYL5
  • Kit and method for detecting methylation level of liver cancer risk gene TSPYL5
  • Kit and method for detecting methylation level of liver cancer risk gene TSPYL5

Examples

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Effect test

Embodiment 1

[0065] Example 1 TSPYL5 Gene methylation fluorescent PCR detection of 163 pairs of liver cancer and paracancerous tissue samples

[0066] Genomic DNA of paraffin samples and frozen tissue samples were extracted using kits from Qiagen (QIAamp DNA FFPE Tissue kit; QIAamp DNA Mini Kit). The concentration and purity of DNA samples were detected by NanoDrop-2000c.

[0067] 300ng DNA sample was digested with methylation-sensitive restriction endonuclease (HinP1I). The digestion system was: DNA 300ng, Buffer (10×) 5μL, methylation-sensitive restriction endonuclease 30U, ddH 2 O supplemented to 50μL, this is the enzyme group. In addition to the treatment without enzyme group, ddH 2 Except for the restriction endonuclease replaced by O, the others are all the same as the enzyme addition group. Negative control (commercial unmethylated human genomic DNA control) and positive control (commercial fully methylated human genomic DNA) were also treated in the same way.

[0068] Design...

Embodiment 2

[0074] Example 2 BSP cloning sequencing method to detect liver cancer and paracancerous tissue TSPYL5 Gene methylation level

[0075] Genomic DNA of paraffin samples and frozen tissue samples were extracted using kits from Qiagen (QIAamp DNA FFPE Tissue kit; QIAamp DNA Mini Kit). The concentration and purity of DNA samples were detected by NanoDrop-2000c.

[0076] 2 μg of DNA samples were modified with sodium bisulfite, and the negative control (commercial unmethylated human genomic DNA control) and positive control (commercial fully methylated human genomic DNA) were also treated in the same way. The specific process is as follows :

[0077] (1) Take 2 μg of DNA and place it in a 2 mL EP tube, add appropriate amount of autoclaved water to make up the volume to 50 μL;

[0078] (2) Add 5.5 μL of freshly prepared 3mol / L NaOH, and bathe in water at 55°C for 20 minutes;

[0079] (3) Add 30 μL of freshly prepared hydroquinone solution (0.04 mol / L) and 520 μL of sodium bisulfi...

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Abstract

The invention discloses a kit and a method for detecting the methylation level of liver cancer risk gene TSPYL5, and belongs to the field of biotechnologies. The kit for detecting the methylation level of the TSPYL5 comprises a primer pair A used for an MSRE-qPCR method or a primer pair B used for a BSP method, wherein the primer sequence is shown in SEQ ID NO. 1-22. According to the kit and the method disclosed by the invention, the relevance between the methylation of the TSPYL5 gene and liver cancer occurrence is found, and the methylation loci of the TSPYL5 are authenticated; the methylation method for the TSPYL5 by virtue of fluorescent quantitative PCR is established by combining specific methylation sensitive restriction endonuclease with the specific primers; the methylation level of the TSPYL5 is detected through the kit and the method based on MSRE-qPCR, the sensitivity is up to 83.9%, and the specificity is up to 93.25%.

Description

[0001] technical field [0002] The invention relates to the field of biotechnology, in particular to a method for detecting liver cancer risk genes TSPYL5 Kits and methods for methylation levels. Background technique [0003] DNA methylation is one of the main contents of epigenetics research. It studies the reversible genetic changes that occur without changing the base sequence of the gene. It is a covalent modification method after DNA molecule replication. In eukaryotes, DNA methylation refers to the transfer of methyl groups to specific bases in DNA molecules using S-adenosylmethionine as a methyl donor under the action of DNA methyltransferases (DNMTs) process, which mainly occurs on cytosines in CpG dinucleotides. In the genome, the regions where the distribution of CpG dinucleotides is relatively concentrated are called CpG islands (CpG islands, CGIs), ranging in size from 100 to 1000 bp, mainly located in the promoter region and the first exon region of genes; th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 郑芳杨国华邱雪平黄一芳邓冠华高嘉嘉
Owner WUHAN UNIV
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